The present invention relates to an improved means and methods of storage and reactivation of entomopathogenic fungal cultures and conidia, especially of the genera Metarhizium.
Insect pathogens are an alternative to the common use of highly toxic chemical insecticides for the control of insect pests. Bacteria such as Bacillus thurigenesis are used with some success as a spray on plants susceptible to infestation with insects such as the gypsy moth. Fungi are another promising group of insect pathogens suitable for use as biological agents for the control of insects. The most common mode of growth and reproduction for fungi is vegetative or asexual reproduction which involves sporulation followed by germination of the spores. Asexual spores, including conidia, form at the tips and the sides of sporogenous cells on the hyphae, the branching filamentous structures of multicellular mycelium. In the proper environment, the conidia germinate, become enlarged,and produce germ tubes. The germ tubes develop, over time, into hyphae which in turn form mycelia. When the insect is inoculated via contact with the living propagules of the entomopathogenic fungi, the infection occurs as soon as the parasite relationship between the fungus and the target insect is established. With the development of infection, the fungus gradually kills the insect.
U.S. Pat. Nos. 5,057,316 and 5,057,315 disclose a method for control and extermination of insects, including roaches, flying insects such as the housefly, and other insects such as the adult form of the corn rootworm. The insects are controlled and exterminated by infection with a fungus that can be pathogenic when administered to the insects in a sufficiently high concentration, by means of an infection chamber. The chamber maintains the spores of a fungus pathogenic to the insects in a viable form, protecting the fungi from the environment (including rain, ultraviolet light and the wind), serves as, or houses, an attractant for the insects, and serves to inoculate the insects with high numbers of spores. The fungal culture provides a continuous supply of spores over a prolonged period of time. The spores attach to the insects and originate germ tubes that penetrate into the insect, which can result in death within three to four days. The chamber design, i.e., shape and color, can be the sole attractants for the insects. Alternatively, food or scents can be used to further enhance the attraction of the insects for the chamber. Although the primary means of infection is by external contact, the insects may also be infected by contact with each other and by ingestion of the spores. In some cases, the ingested fungal conidia can also be toxic.
A historical limitation on the commercial success of insect pathogenic fungi has been their lack of good storage characteristics. Although the chambers described in U.S. Pat. Nos. 5,057,316 and 5,057,315 have been demonstrated to be highly effective both in the laboratory and under actual field test conditions, it is necessary that they withstand shipping and be stable to prolonged storage at room temperature in order to constitute a viable commercial product.
The two most preferred entomopathogenic fungi are Metarhizium anisopliae and Beauveria bassiana. However, only limited success in commercialization of products of these two fungi has been achieved so far because of the obstacles encountered in stabilization, storage, and after-storage reactivation of fungal propagules such as conidia.
There is a series of six stages in the life history of a conidium: formation, maturation, after-ripening, activation, dormancy, and germination. In the dormancy stage, the spore is in a state of reduced physiological activity with an extended period of quiescence, and is most tolerant to the external environmental conditions. There are two types of dormancy: constitutive dormancy involves endogenous constraints that are not overcome simply by supplying conditions suitable for growth; exogenous dormancy is environmentally imposed, and ends when conditions suitable for growth are presented. Exogenous dormancy can be imposed either by withholding a special nutrient required for germination or by the presence of inhibitors, and can be maintained under certain environmental conditions.
It has been known for some time that dried microbial spores, cells, and other type of propagules may be preserved for longer periods of time than fresh ones. However, most scientists accept the generalization that dehydration of living cells may result in massive upheavals of membranes with irreversible loss of the structural and functional integrity (Crowe & Crowe, Stabilization of Membranes in Anhydrobiotic Organisms, In MEMBRANES, METABOLISM AND DRY ORGANISMS, A. C. Leopold, Comstock Publishing Co., Ithica, N.Y., pp. 188-209 (1986); Beker & Rapoport, Conservation of Yeast by Dehydration, In ADVANCES IN BIOCHEMICAL ENGINEERING/BIOTECHNOLOGY, Vol.35 of BIOTECHNOLOGY METHODS, ed. A. Fiechper, Springer Verlag, pp. 127-171 (1987)). This generalization is supported by the fact that there is a significant reduction of germination rate when spores or other types of microbial propagules are dried. Most research work on structural damage caused by dehydration has been focused on yeasts (Beker & Rapoport, 1987). Filamentous fungi produce different types of propagules, and conidium is the one that can be produced abundantly in a short period of time. However, conidia are not tolerant of dehydration. It has been found that conidia of various entomopathogenic fungi are very sensitive to drying processes and lose their viability very quickly (European Patent Application No. 92100010.5 by Eyal, et al.) Efforts have been made to develop mycelium formulations (Australia Patent Application No. P 36 39 508.8 by Andersch, et al.;), however, mycelial formulations are not stable at high temperatures, and have limited applications.
There are several studies on the effect of storage temperature and relative humidity (R.H.) on conidial stability and efficacy of Metarhizium anisopliae and Beauveria bassiana (Daoust & Roberts, Effect of Formulation on the Viability of Metarhizium anisopliae Conidia, J. of Invertebrate Pathology, Vol. 41, pp. 151-160 (1983); Walstad, J. D., "Effects of Environmental Conditions on Two Species of Muscardine Fungi (Beauveria bassiana and Metarhizium anisopliae)", J. of Invertebrate Pathology, Vol. 16, 221-226 (1970)). The results of these studies indicated that conidia of M. anisopliae survived the longest at moderate temperatures in the presence of high R.H. (26.degree. C.+97% R.H. or 19.degree. C.+97% R.H.). However, conidia lost their ability to germinate in a short period of time at 37.degree. C. Germination is the first stage in the establishment of an entomopathogenic fungus.
During transportation and warehouse storage, the temperature could be as high as 37.degree. C. No efforts have been made by the prior art to carefully investigate the status of the conidia that failed to germinate. Although techniques for accelerating and synchronizing germination of conidia of M. anisopliae have been studied by using fresh conidia materials. A period of 10 to 44 hours soaking in distilled water, and a suitable nutrient source are required (Dillon & Charnley, 1985). The conidia of entomopathogenic fungi, such as M. anisopliae and B. bassiana, are hydrophobic in nature. When they are suspended in water, the interface between water and the surface of conidia is characterized by the existence of a high surface tension which prevents the conidia from absorbing water necessary for germination. By carefully examining different surface active agents, it might be possible to alleviate this problem. Surface active agents are characterized by a structure in which the molecule is more or less clearly divided into distinct moieties. One moiety is hydrophilic, and the other hydrophobic. The method employed to quantify the hydrophilic-lipophilic nature of a surface active agent is Hydrophile-Lipophile Balance (HLB) method. In this method, an HLB number is assigned to each surface active agent, and is related by the scale to the suitable application surface active agent. The scale is devised so that the more hydrophilic surface active agents have higher HLB number. Although the HLB method for screening surface active agents has been used to chemical pesticide formulation, the application of HLB method in the screening of suitable surface active agents to reactivate microbial propagules after drying and storage is a new area. Moreover, little is known about the effect of O.sub.2 levels on conidia viability when conidia are stored at different temperature-R.H. combinations. Oxygen is important for the germination of fungal spores.
It is therefore an object of the present invention to provide a method and means for increasing the shelf-life of a pathogenic fungus, especially Metarhizium anisopliae, on a nutrient medium.
It is a further object of the present invention to provide improved packaging for insect infection chambers utilizing a pathogenic fungus.
It is another object of the present invention to provide methods for storage to increase the shelf life of conidia of entomopathogenic fungi, at a higher temperature.
It is a further object of the present invention is to develop a suitable method to reactivate the conidia after storage.